Biochemistry 1986, 25,4219-4223
4219
A Spectral Study of Cobalt(11)-Substituted Bacillus cereus Phospholipase Ct Roy Bicknell,* Graeme R. Hanson,t and Barton Holmquist Centerfor Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 021 15 Clive Little Institute of Medical Biology, University of Tromso, N-9000 Tromso, Norway Received January 30, 1986; Revised Manuscript Received April 3, 1986
The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(I1) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(I1) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A X . , & Mannervik, B. (1983) J . Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S . (1984) Biochemistry 23, 685-6891, transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S . (1974) Biochemistry 13, 1160-11691, and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-53561, Phospholipase C is so far unique in having two such sites. ABSTRACT:
Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus contains two zinc ions (Little & Otnaess, 1975). Studies have shown that one of these is exchange-labile and catalytically essential while the other is more tightly bound and appears to have a structural role (Little & Otnaess, 1975; Little, 1981). Several metal ions, including the spectroscopic probe cobalt(II), can be substituted for zinc to give enzymes of reduced activity and altered substrate specificity (Little & Otnaess, 1975; Little et al., 1982). However, the structural characteristics of the metal binding sites have not been previously investigated, although the zinc ions are known from X-ray crystallography to be separated by around 6 A (Aalmo et al., 1984). This paper presents a detailed spectral study of the cobalt(I1)-substituted enzyme. The spectra suggest that Co(I1) ions-and by inference zinc ions-occupy octahedral coordination sites and that the coordination environments of the two ion sites are remarkably similar. MATERIALS AND METHODS Materials. Phospholipase C was isolated from cultures of Bacillus cereus as described by Myrnes and Little (1980). Phospholipase C concentrations were determined from Azso where E = 51 000 M-' cm-' (Little, 1977). [Co,Zn(PLC)]' was prepared by extensive dialysis of [Zn,Zn(PLC)] against solutions of cobalt(I1) chloride (2 X M) at pH 7.3 (Little, 197 1). [-,Co(PLC)] and [Co,Co(PLC)] were prepared by first making apoenzyme (Little, 1977) and then either adding 0.9 mol of Co(II)/mol of apoenzyme (for [-,Co(PLC)]) or dialyzing apoenzyme against solutions of cobalt(I1) chloride 'This work was supported by funds for Research and Teaching in the CBBSM. R.B. holds an SERC/NATO British Overseas Postdoctoral Fellowship. G.R.H. was supported during the EPR study by National Institutes of Health Grant RR-01008 awarded to the National Biomedical ESR Center, University of Wisconsin, Milwaukee, WI. * Author to whom correspondence should be addressed. 'Present address: Department of Physics, Monash University, Clayton, Victoria, Australia.
(2 X M). Enzyme samples were concentrated by ultrafiltration, free Co(I1) being removed by repeated concentration of enzyme followed by dilution in 0.1 M sodium acetate (pH 6.0). Free Co(I1) represented
